Many of the most clinically important chemotherapeutic agents inhibit the metabolism of tumor cells. Our overarching goal is to develop a complete and quantitative understanding of the metabolic differences between normal and cancer cells, and to use this knowledge to guide the rational design of novel anticancer regimens. To achieve this goal, we are developing methods that apply state-of-the-art liquid chromatography- electrospray ionization-triple quadrupole mass spectrometry technology to probe cellular metabolism in a dynamic, quantitative, and comprehensive manner. To date, we have succeeded in developing methods for measuring metabolite concentrations and fluxes from several microbes. Here we propose to extend these methods to enable reliable measurement of metabolite concentrations and fluxes in normal and cancer cells. Specifically, we aim to enable quantitation of the concentrations of at least 150 different known, structurally-defined intracellular metabolites. We also aim to measure, using isotopic tracers, the fluxes through central carbon, lipid, amino acid, and nucleotide metabolism. We will apply the analytical technology that we develop to map major metabolic differences between normal and cancer cells and to study the dynamic response of these cells to treatment with anti-metabolite anticancer drugs. The methods developed here will have long-term value for understanding the mechanism of action (and toxicity) of anti-metabolite anticancer drugs, for characterizing the metabolic differences between drug-responsive and resistant cancer cells, and for suggesting new approaches to inhibiting metabolism that will specifically kill cancer cells.